PRRSV GP5蛋白在杆状病毒表达系统中的表达
|
摘要
参照包含猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV) VR2332株完整ORF5基因的载体pMD18T-ORF5的核酸序列设计引物,扩增全长的PRRSV ORF5基因片段,同时以SOE-PCR技术扩增缺失信号肽和跨膜区的ORF5核酸片段ORF5NC,分别克隆至Bac-to-Bac杆状病毒表达系统的供体质粒pFastBac-HTA,获得重组转移载体pFastBacHTA-ORF5及截短表达的重组转移载体pFastBacHTA-ORF5NC。将供体质粒分别转化DH10Bac细胞,获得重组穿梭质粒,转染Sf9昆虫细胞,获得重组杆状病毒。SDS-PAGE和Western blot检测结果表明,在Sf9细胞中分别成功表达了全长的GP5重组蛋白和缺失信号肽、跨膜区的截短GP5重组蛋白。2种重组蛋白经纯化后,免疫小鼠制备抗血清,ELISA方法检测免疫GP5全长和截短重组蛋白的小鼠血清中抗体滴度分别为1∶800和1∶1 600,表明重组蛋白具有良好的免疫原性。本试验为进一步分析重组蛋白诱导中和抗体产生的能力以及为PRRSV基因工程亚单位疫苗的研究奠定基础。
To express glycoprotein 5 of porcine reproductive and respiratory syndrome virus in baculovirus expression system and analyze the immunogenicity of the expressed product,the primers were designed according to the nucleotide sequence of vector pMD18 T-ORF5 containing ORF5 gene of PRRSV VR2332 strain.Complete ORF5 gene and incomplete ORF5 gene lack of transmembrane domain and signal peptide were amplified by PCR and the two fragments were respectively cloned into the pFast-HTA vector to obtain two recombinant vector pFastBacHTA-ORF5 and pFastBacHTA-ORF5 NC,and then transformed into DH10-Bac cells that contain baculovirus shuttle plasmid.Recombinant transfer vector pFastBacHTA-ORF5 and truncated expressed recombinant transfer vector pFastBacHTA-ORF5 NC were obtained and then respectively transfected into Sf9 cells to obtain two recombinant baculovirus.SDS-PAGE and Western blot result showed that the full-length GP5 recombinant protein and the deletion signal peptide and the truncated GP5 recombinant protein in the transmembrane region were successfully expressed in Sf9 cells,respectively.Mice were immunized with purified recombinant proteins to prepare antiserum,the titer of antiserum were detected by indirect enzyme linked immunosorbent assay(ELISA) with purified PRRSV as coating antigen,the titer of the full length GP5 antiserum was 1∶800 and 1∶1 600 for truncated GP5 antiserum,indicating that the recombinant protein has good immunogenicity.This study laid a foundation for the further analysis of the ability of recombinant protein to induce neutralizing antibody production and for the study of PRRSV gene engineering subunit vaccine.
To express glycoprotein 5 of porcine reproductive and respiratory syndrome virus in baculovirus expression system and analyze the immunogenicity of the expressed product,the primers were designed according to the nucleotide sequence of vector pMD18 T-ORF5 containing ORF5 gene of PRRSV VR2332 strain.Complete ORF5 gene and incomplete ORF5 gene lack of transmembrane domain and signal peptide were amplified by PCR and the two fragments were respectively cloned into the pFast-HTA vector to obtain two recombinant vector pFastBacHTA-ORF5 and pFastBacHTA-ORF5 NC,and then transformed into DH10-Bac cells that contain baculovirus shuttle plasmid.Recombinant transfer vector pFastBacHTA-ORF5 and truncated expressed recombinant transfer vector pFastBacHTA-ORF5 NC were obtained and then respectively transfected into Sf9 cells to obtain two recombinant baculovirus.SDS-PAGE and Western blot result showed that the full-length GP5 recombinant protein and the deletion signal peptide and the truncated GP5 recombinant protein in the transmembrane region were successfully expressed in Sf9 cells,respectively.Mice were immunized with purified recombinant proteins to prepare antiserum,the titer of antiserum were detected by indirect enzyme linked immunosorbent assay(ELISA) with purified PRRSV as coating antigen,the titer of the full length GP5 antiserum was 1∶800 and 1∶1 600 for truncated GP5 antiserum,indicating that the recombinant protein has good immunogenicity.This study laid a foundation for the further analysis of the ability of recombinant protein to induce neutralizing antibody production and for the study of PRRSV gene engineering subunit vaccine.
引文
[1] 郭宝清,陈章水,刘文兴,等.从疑似PRRS流产胎儿分离PRRSV的研究[J].中国畜禽传染病,1996,87(2):1-4.
[2] LI Y,WANG X,BO K,et al.Emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus in the Mid-Eastern region of China[J].Vet J,2007,174(3):577-584.
[3] XU X G,WANG Z S,ZHANG Q,et al.Baculovirus as a PRRSV and PCV2 bivalent vaccine vector:baculovirus virions displaying simultaneously GP5 glycoprotein of PRRSV and capsid protein of PCV2[J].J Virol Methods,2012,179(2):359-366.
[4] ZHENG Q,CHEN D,LI P,et al.Co-expressing GP5 and M proteins under different promoters in recombinant modified vaccinia virus Ankara(rMVA)-based vaccine vector enhanced the humoral and cellular immune responses of porcine reproductive and respiratory syndrome virus(PRRSV)[J].Virus Gene,2007,35(3):585-595.
[5] PRIZADEH B,DEA S.Monoclonal antibodies to the ORF5 product of porcine reproductive and respiratory syndrome virus define linear neutralizing determinants[J].J Gen Virol,1997,78(8):1867-1873.
[6] OSTROWSKI M,GALEOTA J A,JAR A M,et al.Identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus GP5 ectodomain[J].J Virol,2002,76(9):4241-4250.
[7] GONIN P,PIRZADEH B,GAGNON C A,et al.Seroneutralization of porcine reproductive and respiratory syndrome virus correlates with antibody response to the GP5 major envelope glycoprotein[J].J Vet Diagn Invest,1999,11(1):20-26.
[8] 李素萍.PRRSV重组N蛋白ELISA检测方法的建立及单克隆抗体的制备[D].河南郑州:河南农业大学,2009.
[9] LUNNEY J K,FANG Y,LADINIG A,et al.Porcine reproductive and respiratory syndrome virus (prrsv):pathogenesis and interaction with the immune system[J].Ann Rev Anim Biosci,2016(4):129-154.
[10] PLANA D J,CLIMENT I,SARRASECA J,et al.Baculovirus expression of proteins of porcine reproductive and respiratory syndrome virus strain Olot/91.Involvement of ORF3 and ORFS proteins in protection[J].Virus Gene,1997,14(1):19-29.
[11] MUSIC N,GAGNON C A.The role of porcine reproductive and respiratory syndrome (PRRS) virus structural and non-structural proteins in virus pathogenesis[J].Anim Health Res Rev,2010,11(2):135-163.
[12] 谷红,杨汉春,郭鑫,等.PRRSV BJ24株ORF5基因的原核表达与重组蛋白的纯化[J].畜牧兽医学报,2004,35(1):64-69.
[13] MENA J A,KAMEN A A.Insect cell technology is a versatile and robust vaccine manufacturing platform[J].Exp Rev Vaccines,2011,10(7):1063-1081.
[14] ALNEMRI E S,LITWACK G.The steroid binding domain influences intracellular solubility of the baculovirus overexpressed glucocorticoid and mineralocorticoid receptors[J].Biochemistry,1993,32(20):5387-5393.
[15] AILOR E,BETENBAUGH M J.Overexpression of a cytosolic chaperone to improve solubility and secretion of a recombinant IgG protein in insect cells[J].Biotechnol Bioeng,1998,58(2/3):196-203.
[16] KIM P S,ARVAN P.Calnexin and BiP act as sequential molecular chaperones during thyroglobulin folding in the endoplasmic reticulum[J].J Cell Biol,1995,128(1/2):29-38.
[2] LI Y,WANG X,BO K,et al.Emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus in the Mid-Eastern region of China[J].Vet J,2007,174(3):577-584.
[3] XU X G,WANG Z S,ZHANG Q,et al.Baculovirus as a PRRSV and PCV2 bivalent vaccine vector:baculovirus virions displaying simultaneously GP5 glycoprotein of PRRSV and capsid protein of PCV2[J].J Virol Methods,2012,179(2):359-366.
[4] ZHENG Q,CHEN D,LI P,et al.Co-expressing GP5 and M proteins under different promoters in recombinant modified vaccinia virus Ankara(rMVA)-based vaccine vector enhanced the humoral and cellular immune responses of porcine reproductive and respiratory syndrome virus(PRRSV)[J].Virus Gene,2007,35(3):585-595.
[5] PRIZADEH B,DEA S.Monoclonal antibodies to the ORF5 product of porcine reproductive and respiratory syndrome virus define linear neutralizing determinants[J].J Gen Virol,1997,78(8):1867-1873.
[6] OSTROWSKI M,GALEOTA J A,JAR A M,et al.Identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus GP5 ectodomain[J].J Virol,2002,76(9):4241-4250.
[7] GONIN P,PIRZADEH B,GAGNON C A,et al.Seroneutralization of porcine reproductive and respiratory syndrome virus correlates with antibody response to the GP5 major envelope glycoprotein[J].J Vet Diagn Invest,1999,11(1):20-26.
[8] 李素萍.PRRSV重组N蛋白ELISA检测方法的建立及单克隆抗体的制备[D].河南郑州:河南农业大学,2009.
[9] LUNNEY J K,FANG Y,LADINIG A,et al.Porcine reproductive and respiratory syndrome virus (prrsv):pathogenesis and interaction with the immune system[J].Ann Rev Anim Biosci,2016(4):129-154.
[10] PLANA D J,CLIMENT I,SARRASECA J,et al.Baculovirus expression of proteins of porcine reproductive and respiratory syndrome virus strain Olot/91.Involvement of ORF3 and ORFS proteins in protection[J].Virus Gene,1997,14(1):19-29.
[11] MUSIC N,GAGNON C A.The role of porcine reproductive and respiratory syndrome (PRRS) virus structural and non-structural proteins in virus pathogenesis[J].Anim Health Res Rev,2010,11(2):135-163.
[12] 谷红,杨汉春,郭鑫,等.PRRSV BJ24株ORF5基因的原核表达与重组蛋白的纯化[J].畜牧兽医学报,2004,35(1):64-69.
[13] MENA J A,KAMEN A A.Insect cell technology is a versatile and robust vaccine manufacturing platform[J].Exp Rev Vaccines,2011,10(7):1063-1081.
[14] ALNEMRI E S,LITWACK G.The steroid binding domain influences intracellular solubility of the baculovirus overexpressed glucocorticoid and mineralocorticoid receptors[J].Biochemistry,1993,32(20):5387-5393.
[15] AILOR E,BETENBAUGH M J.Overexpression of a cytosolic chaperone to improve solubility and secretion of a recombinant IgG protein in insect cells[J].Biotechnol Bioeng,1998,58(2/3):196-203.
[16] KIM P S,ARVAN P.Calnexin and BiP act as sequential molecular chaperones during thyroglobulin folding in the endoplasmic reticulum[J].J Cell Biol,1995,128(1/2):29-38.